目的 建立一种无气管插管并不使用呼吸机通气的小鼠微创主动脉弓缩窄(TAC)技术.方法 随机将40只雄性2～4个月龄C57BL/6小鼠分为TAC手术组和假手术组,每组20只.TAC手术组小鼠用戊巴比妥钠溶液腹膜内注射麻醉,实施胸骨部分切开术后暴露主动脉弓,然后将缝线穿过主动脉弓下方并将主动脉弓系在钝的弯曲成90°的27号针上,完成结扎后拔出27号针.假手术组操作过程同TAC手术组,但不进行结扎.对比分析两组小鼠心脏质量/体重比、心肌肥大标志基因表达以及在体心脏功能.结果 与假手术组相比,TAC手术组小鼠在术后第28天心脏质量/体重比显著升高[(6.21±0.20)mg/g vs(3.94±0.19)mg/g,P<0.01],心肌肥大标志基因心房钠尿肽和脑钠肽表达升高(3.48±0.37 vs 1.00±0.12、3.92±0.41 vs 1.00±0.15,P均<0.01),左心室射血分数和左心室短轴缩短率降低[(40.18±1.96)％vs(53.26±2.32)％、(23.12±2.08)％vs(32.16±2.64)％,P均<0.01].手术过程中及术后28 d内小鼠存活率为95.00％(38/40).结论 微创TAC手术制备心肌肥厚模型小鼠的死亡率低,并能高效诱导心肌肥厚和降低心功能.

Estrogen receptor beta (ERβ) has been shown to play a therapeutic role in inflammatory bowel disease (IBD). However, the mechanism underlying how ERβ exerts therapeutic effects and its relationship with P2X3 receptors (P2X3R) in rats with inflammation is not known. In our study, animal behavior tests, visceromotor reflex recording, and Western blotting were used to determine whether the therapeutic effect of ERβ in rats with inflammation was related with P2X3R. In complete Freund adjuvant (CFA)-induced chronic inflammation in rats, paw withdrawal threshold was significantly decreased which were then reversed by systemic injection of ERβ agonists, DPN or ERB-041. In 2,4,6-trinitrobenzene sulfonic acid (TNBS)-induced colitis in rats, weight loss, higher DAI scores, increased visceromotor responses, and inflammatory responses were reversed by application of DPN or ERB-041. The higher expressions of P2X3R in dorsal root ganglia (DRG) of CFA-treated rats and those in rectocolon and DRG of TNBS-treated rats were all decreased by injection of DPN or ERB-041. DPN application also inhibited P2X3R-evoked inward currents in DRG neurons from TNBS rats. Mechanical hyperalgesia and increased P2X3 expression in ovariectomized (OVX) CFA-treated rats were reversed by estrogen replacements. Furthermore, the expressions of extracellular signal-regulated kinase (ERK) in DRG and spinal cord dorsal horn (SCDH) and c-fos in SCDH were significantly decreased after estrogen replacement compared with those of OVX rats. The ERK antagonist U0126 significantly reversed mechanical hyperalgesia in the OVX rats. These results suggest that estrogen may play an important therapeutic role in inflammation through down-regulation of P2X3R in peripheral tissues and the nervous system, probably via ERβ, suggesting a novel therapeutic strategy for clinical treatment of inflammation.

Objective To investigate the regulatory effect of protein kinase A(PKA) and protein kinase C(PKC) on the function of ATP receptor P2X3 in cultured dorsal root ganglion(DRG) neurons.Methods Rat DRG neurons were collected,cultured and subjected to exogenous ATP treatment to induce transient inward current.Whole-cell patch clamp recording was employed to observe the effect of TNP-ATP(P2X3 and P2X2/3 receptor antagonist) on the induced current.Meanwhile,the regulatory effects of PKA and PKC agonist on ATP-induced transient inward current were also observed.Results TNP-ATP inhibited ATP-induced transient current in cultured DRG neurons in a dose-dependent manner,with the IC50 value being(21.7 ± 7.6) nmol/L.PKA agonist forskolin(1 μmol/L) and PKC agonist PMA(1 μmol/L) inhibited ATP-induced transient current rapidly and reversibly.Conclusion PKA and PKC can inhibit ATP-induced transient inward current in cultured DRG neurons,probably through inhibiting the function of P2X3 receptors.It indicates that P2X3 receptor regulation by protein kinases may participate in the development of pain.

Objective To investigate the modulatory effect of progesterone on peripheral pain.Methods Female SD rats were divided into four groups,namely,a vehicle group,a progesterone(500 μg) group,an RU38486(20 μg) group and a progesterone(500 μg) plus RU38486(20 μg) group.Von-Frey hair test was adopted to determine the peripheral mechanical pain thresholds at the following 3 time points: preinjection,30 min and 60 min after subcutaneous injection at the neck.Local effect of progesterone was also tested in SD rats by Von-Frey hair test 15 min after plantar injection of progesterone(25 μg)and normal saline(control).Results The peripheral mechanical pain threshold was significantly increased 30 min after subcutaneous injection of progesterone at the neck compared with the vehicle group,but the threshold was not significantly different 60 min after injection.RU38486 injection at the neck significantly decreased the pain threshold of plantar at 30 min and 60 min after injection,and the peripheral mechanical pain threshold was not significantly altered at 30 min and 60 min after co-injection of progesterone and RU38486.RU38486 could block the pain inhibiting effect of progesterone. The peripheral mechanical pain threshold was also increased 15 min after plantar injection of progesterone compared with the control group.Conclusion Our findings suggest that progesterone can inhibit the peripheral mechanical pain,which might be mediated by progesterone receptor.

There is evidence that gonadal hormones may affect the perception of painful stimulation, although the underlying mechanisms remain unclear. This investigation was undertaken to determine whether the adenosine 5′-triphosphate (ATP) receptor subunit, P2X3, is involved in the modulatory action of estrogen in peripheral pain signal transduction in dorsal root ganglion (DRG). The mechanical pain behavior test, real-time quantitative reverse transcription–polymerase chain reaction analysis, and Western blot methods were used to determine the mean relative concentrations and functions of P2X3 receptors in DRG in sham, ovariectomized (OVX), and estradiol replacement (OVX+E2) female rats and in sham and orchiectomized male rats. The mechanical hyperalgesia appeared after ovariectomy, which was subsequently reversed after estradiol replacement, whereas it was not observed after orchiectomy in male rats. Plantar injection of 2′(3′)-O-(2,4,6-trinitrophenyl) ATP (TNP-ATP), a P2X3 and P2X2/3 receptor antagonist, resulted in an increase of the pain threshold force in OVX rats while had no effect on sham rats. Furthermore, A-317491, a selective P2X3/P2X2/3 receptor antagonist, significantly reversed the hyperalgesia of OVX rats. Injection of ATP into the plantars also caused a significant increase of the paw withdrawal duration in OVX rats compared with that seen in the sham group, which became substantially attenuated by TNP-ATP. P2X3 receptors expressed in DRG were significantly increased in both mRNA and protein levels after ovariectomy and then reversed after estrogen replacement, while a similar increase was not observed after orchiectomy in male rats. Furthermore, P2X3 mRNA was significantly decreased 24 h after the application of 17β-estradiol in a concentration-dependent manner in cultured DRG neurons. ICI 182,780, an estrogen receptor antagonist, blocked the reduction in the protein level. These results suggest that the female gonadal hormone, 17β-estradiol, might participate in the control of peripheral pain signal transduction by modulating P2X3 receptor-mediated events in primary sensory neurons, probably through genomic mechanisms.

目的 研究母鼠怀孕中后期地塞米松(DEX)暴露对子代大鼠发育过程中痛觉敏感性的影响.方法 以母鼠孕期中后半程(9～12 d)注射DEX(每天100 μg/kg)和注射生理盐水后出生的大鼠为样本,通过足底部痛阈测定,观察子代大鼠出生后3 d及1、2、3、4、5、6、7、8周时机械痛阈和热痛阈的变化.结果 两组子代大鼠的外周机械痛阈和热痛阈均随着年龄的增长逐渐增加.出生后3 d和1周时,DEX组机械痛阈显著低于对照组(P<0.05),而2周和3周时,DEX组机械痛阈明显著高于对照组(P<0.05),4周以后两组间无显著差别.对于热痛阈,大鼠出生后3 d DEX组显著低于对照组(P<0.05),而出生后1周及以后的时期两组间无显著差异.结论 母体孕中后期注射DEX可以增强子代大鼠发育早期的痛觉敏感性,而这种影响可能是短暂性的.

Background: Progesterone has been shown to play a role in pain perception. However, the effects of progesterone on P2X3 receptors, the nociception-related receptors in primary sensory neurons, remain unclear. Methods: We investigated the effects of progesterone on P2X3-receptor-mediated responses of rat dorsal root ganglion neurons by using whole-cell patch clamp techniques. Results: Progesterone (10 pmol/l to 1 µmol/l) inhibited ATP (100 µmol/l)-induced transient currents and the transient component of biphasic currents in a dose-dependent manner, but had no effect on the sustained currents. The effect of progesterone could be blocked by its receptor inhibitor, RU38486. Actidione, an inhibitor of protein synthesis, did not change the rapid effect. Investigations of the signaling pathways of progesterone showed that a nonselective antagonist of protein kinase, H-9, totally blocked the depressive effect of progesterone on ATP currents in a dose-dependent manner. Blocking protein kinase A with H-89 or KT5720 also affected progesterone-induced depression. The protein kinase A activator, forskolin, mimicked the effect of progesterone on ATP currents. Conclusion: These results suggest that progesterone may modulate pain signal transmission on dorsal root ganglia via regulating P2X3 receptor function. The cAMP-PKA signaling pathway is involved in the downregulating effect of progesterone on P2X3 receptors.

The purpose of this study was to establish a model of trigeminal neuralgia (TN) through an approach from lower edge of cheekbone and to observe the functional changes in the voltage-gated potassium currents in the cultured trigeminal ganglion (TG) neurons. Thirty Sprague-Dawley male rats were divided into two groups, the sham-operated (sham) group and the operated group. The TN model was carried out by using a chronic constriction injury of the infraorbital nerve (ION-CCI) from lower edge of cheekbone. Peripheral pain threshold test and whole-cell patch clamp recording were used to determine the difference between sham and ION-CCI rats. The withdrawal threshold of whisker pad in operated side of ION-CCI rat was decreased significantly from 6 d after operation and then maintained until 21 d, with the lowest on the 15th day. The threshold of whisker pad in non-operated side of operated rats was also decreased significantly compared with that in the sham group. Delayed rectifier potassium current (I(K)) in cultured ION-CCI TG neurons was decreased significantly compared with that in the sham group. Transient outward potassium currents (I(A)) in both operated and non-operated sides of TG neurons from ION-CCI rats were also reduced significantly compared with that in the sham group. The present study provided a new method of ION-CCI. In this model, the decrease of I(A) and I(K) might contribute, at least in part, to the decrease in mechanical pain threshold of whisker pad and the subsequent hyperalgia.

孕酮(progesterone,PROG)不仅存在于生殖系统,而且在神经系统也有合成.孕酮受体在中枢和外周神经系统中均有分布,参与神经系统的各项功能,其中包括对疼痛的调节.孕酮及其代谢产物对生理性痛和炎性痛均有抑制效应,孕酮对雌激素介导的外周痛觉增敏也有抑制效应.另一方面,孕酮增强神经病理性痛的痛觉异常和痛觉过敏.孕酮可以通过调节某些痛觉相关的神经递质受体的表达和功能以及影响疼痛下行抑制通路,从而完成对痛觉调节.

目的:观察结直肠三硝基苯磺酸(TNBS)致炎条件下,改变大鼠体内雌激素和孕激素水平对背根神经节(DRG)中P2X3和P2X2 mRNA表达的影响.方法:SD大鼠分为4组:假手术组、去卵巢组、去卵巢加雌激素替代组、去卵巢加雌孕激素替代组,每组手术完成后饲养5周,再行TNBS致炎,5～7 d后用于实验.实时定量RT-PCR法检测各组P2X3 和P2X2 mRNA的表达.结果:去卵巢组大鼠DRG中P2X3 mRNA表达明显低于假手术组,差异有统计学意义(1.08±0.35 vs 1.66±0.66,P<0.05,n=4),雌激素替代组有明显上升(2.06±0.58),明显高于去卵巢组(P<0.01,n=5),而雌孕激素同时替代组明显低于单独添加雌激素组(2.06±0.58 vs 0.87±0.28,P<0.01,n=5).各组大鼠DRG中P2X2 mRNA表达无统计学差异.结论:结直肠TNBS致炎条件下,雌激素可促进大鼠DRG中P2X3 mRNA表达,但对P2X2 mRNA无明显影响;孕激素的作用可能与雌激素正好相反.炎性条件下,雌孕激素可能通过影响P2X3受体的表达发挥调节炎性痛作用.

P2X(3) and P2X(2/3) receptors are expressed in peripheral tissues and dorsal root ganglia (DRG) and participate in peripheral pain. However, the mechanisms underlying P2X receptor-mediated nociception at different ovarial hormone levels has not been examined. In this study, 24 female rats were randomly divided into sham-operated (sham), ovariectomized (OVX), estrogen-treated, and estrogen-progesterone-treated groups with colitis. In each group, the visceromotor reflex (VMR) to colorectal distension was tested and the DRG were harvested for a real-time PCR analysis of P2X(3) and P2X(2) receptor mRNA. In OVX rats with colitis we found that the VMR to colorectal distension and P2X(3) receptor mRNA in DRG were both significantly decreased. Estrogen replacement reversed the decrease. However, neither the VMR nor the P2X(3) mRNA level in DRG from OVX colitis rats was reversed by the complex of estrogen and progesterone. Patch-clamp recording showed that in colitis rats, estradiol rapidly potentiated the sustained and transient currents evoked by ATP to 336+/-49% and 122+/-12% of controls, respectively, in a subpopulation of DRG neurons, which were blocked by ICI 182, 780, an antagonist of the estrogen receptor. Whereas progesterone rapidly inhibited the transient currents induced by ATP to 67+/-10% of control and had no effect on the sustained currents evoked by the same agonist. These results indicate that P2X(3) receptors are likely to be an important contributor to the altered colonic functions in colitis rats, where the underlying mechanisms are closely related to endogenous estrogen modulation.

目的:观察结直肠三硝基苯磺酸(TNBS)致炎条件下,改变大鼠体内雌激素和孕激素水平对背根神经节(DRG)中P2X3和P2X2mRNA表达的影响。方法:SD大鼠分为4组:假手术组、去卵巢组、去卵巢加雌激素替代组、去卵巢加雌孕激素替代组,每组手术完成后饲养5周,再行TNBS致炎,5～7d后用于实验。实时定量RT-PCR法检测各组P2X3和P2X2mRNA的表达。结果:去卵巢组大鼠DRG中P2X3mRNA表达明显低于假手术组,差异有统计学意义(1.08±0.35vs1.66±0.66,P<0.05,n=4),雌激素替代组有明显上升(2.06±0.58),明显高于去卵巢组(P<0.01,n=5),而雌孕激素同时替代组明显低于单独添加雌激素组(2.06±0.58vs0.87±0.28,P<0.01,n=5)。各组大鼠DRG中P2X2mRNA表达无统计学差异。结论:结直肠TNBS致炎条件下,雌激素可促进大鼠DRG中P2X3mRNA表达,但对P2X2mRNA无明显影响;孕激素的作用可能与雌激素正好相反。炎性条件下,雌孕激素可能通过影响P2X3受体的表达发挥调节炎性痛作用。

To elucidate the pharmacological profile of P2X receptors and the probable expression of P2Y receptors in otic ganglion neurons from 17-day-old rats, single neurons were enzymatically isolated and maintained in tissue culture for up to 30 h. Whole-cell voltage-clamp recording was carried out at a holding potential of -60 mV. Most otic ganglion neurons responded to adenosine 51-triphosphate (ATP), 2-methylthio ATP (2MeSATP) and alpha, beta-methylene ATP (up-meATP) with sustained currents and EC50 values of 19 mu M, 47 mu M and 94 mu M, respectively. 2',3'-O-trinitrophenyl-ATP (TNP-ATP) inhibited the response to alpha beta-meATP and ATP with an IC50 values of 3.9 nM and 18.3 nM, respectively, which was closed to that observed in nodose neurons. The response to ATP was antagonized by suramin and cibacron blue. The dose-response curve of suramin against ATP response at a pH of 6.5 was shifted to the left compared to that at a pH of 7.4. Diinosine pentaphosphate (Ip(5)I), which blocks P2X(3), but not P2X(2/3)-mediated responses, had no effect on the currents evoked by ATP or alpha beta-meATP. In some neurons, uridine 5'-triphosphate (UTP) induced a tiny, but long-lasting current with a mean amplitude of 0.034 +/- 0.011 nA. Reverse transcriptase-polymerase chain reaction (RT-PCR) confirmed the expression of mRNAs for P2X(2), P2X(3), P2X(4), P2X(6) and P2X(7), but not for P2X(1) and P2X(5) receptors in otic ganglion. In conclusion, in rat otic ganglion neurons, P2X(2/3) heteromultimer receptors dominate, but P2X(7) and P2Y(2) or P2Y(4) receptors also play roles. (C) 2008 Elsevier Inc. All rights reserved.

P2X3 and P2X2/3 receptors appear to be expressed in peripheral tissues and dorsal root ganglia (DRG) and participate in producing peripheral pain. However, the mechanisms underlying the P2X receptors mediated nociception in the different ovarial hormone levels remain unclear. We found that after ovariectomy, the peripheral mechanical allodynic and nocifensive behavioral responses were developed, which could be reversed by P2X receptor antagonists, while the visceromotor reflex to colorectal distention was decreased, with the longer latency and higher threshold. Immunohistochemial studies revealed that P2X3 expression was significantly higher in small and medium dorsal root ganglion neurons. Real time PCR results showed that the P2X3 mRNA was in a higher level in both dorsal root ganglion neurons and rectocolon. Patch‐clamp recording results showed an increased number of cells responded to ATP with a transient current which was mediated by P2X3 receptors, while no significant change in the amplitude of current. These results indicate P2X3 is likely an important mechanism contributing to the altered receptor functions in DRG and rectocolon after ovariectomy. (Supported by NSFC No: 30570597 and program of Changjiang scholar and innovative team)

Objective To investigate the brain water content in murine model with central nervous system (CNS) cryptococcal infection.Methods Mice were randomly devided into test group and control group. Test group mice were injected with Cryptococcus neoformans via the tail vein,and control group mice received saline. Brain water content of murine model was measured at 6 h,12 h,24 h,48 h,72 h respectively.Results Compared with the control group,brain water content in the test group began to rise at 6h and reached maximum at 24h.Conclusions Mouse model of cryptococcal meningitis with brain edema was successfully established.

Objective:To investigate the modulatory effect of gonadal hormone on peripheral pain.Methods:Orchiectomized and ovariectomized rats models were established.Radio heater and Von-Frey hair were used to determine the peripheral mechani- cal and thermal pain threshold in gonadectomized rats and their corresponding sham-controls.Results:The body weight was in- creased and the uterus weight was decreased after ovariectomy in female rats(P0.01).There was no significant change in the body weight of male rats after orchiectomization.The 100% hind paw withdrawal threshold to Von-Frey hair stimulation was decreased significantly after ovariectomization in female rats,with no significant change in the thermal pain threshold.There was no significant changes in the 100% hind paw withdrawal threshold after Von-Frey hair stimulation or thermal pain threshold af- ter orchidectomy in male rats.Conclusion.These results suggest that changes in androgen level of male rats have no influence on the peripheral basal pain threshold,and female gonadal hormone may inhibit the peripheral pain signaling and has no effect on thermal pain threshold.